RESUMO
Background: Although the functional redundancy of catechol 1,2-dioxygenase (C12O) genes has been reported in several microorganisms, limited enzymes were characterised, let alone the advantage of the coexistence of the multiple copies of C12O genes. Results: In this study, four novel C12O genes, designated catA, catAI, catAII and catAIII, in the naphthalene-degrading strain Pseudomonas putida ND6, were cloned and characterised. Phylogenetic analysis of their deduced amino acid sequences revealed that the four C12O isozymes each formed independent subtrees, together with homologues from other organisms. All four enzymes exhibited maximum activity at pH 7.4 and higher activity in alkaline than in acidic conditions. Furthermore, CatA, CatAI and CatAIII were maximally active at a temperature of 45°C, whereas a higher optimum temperature was observed for CatAII at a temperature of 50°C. CatAI exhibited superior temperature stability compared with the other three C12O isozymes, and kinetic analysis indicated similar enzyme activities for CatA, CatAI and CatAII, whereas that of CatAIII was lower. Significantly, among metal ions tested, only Cu2+ substantially inhibited the activity of these C12O isozymes, thus indicating that they have potential to facilitate bioremediation in environments polluted with aromatics in the presence of metals. Moreover, gene expression analysis at the mRNA level and determination of enzyme activity clearly indicated that the redundancy of the catA genes has increased the levels of C12O. Conclusion: The results clearly imply that the redundancy of catA genes increases the available amount of C12O in P. putida ND6, which would be beneficial for survival in challenging environments.
Assuntos
Pseudomonas putida/enzimologia , Pseudomonas putida/genética , Catecol 1,2-Dioxigenase/genética , Temperatura , Biodegradação Ambiental , Clonagem Molecular , Catecol 1,2-Dioxigenase/análise , Catecol 1,2-Dioxigenase/metabolismo , Genes Bacterianos , Concentração de Íons de Hidrogênio , Isoenzimas , MetaisRESUMO
Fifteen actinomycete strains were evaluated for their potential use in removal of polycyclic aromatic hydrocarbons (PAH). Their capability to degrade of naphthalene, phenanthrene, and pyrene was tested in minimal medium (MM) and MM with glucose as another substrate. Degradation of naphthalene in MM was observed in all isolates at different rates, reaching maximum values near to 76% in some strains of Streptomyces, Rhodococcus sp. 016 and Amycolatopsis tucumanensis DSM 45259. Maximum values of degradation of phenanthrene in MM occurred in cultures of A. tucumanensis DSM 45259 (36.2%) and Streptomyces sp. A12 (20%), while the degradation of pyrene in MM was poor and only significant with Streptomyces sp. A12 (4.3%). Because of the poor performance when growing on phenanthrene and pyrene alone, Rhodococcus sp. 20, Rhodococcus sp. 016, A. tucumanensis DSM 45259, Streptomyces sp. A2, and Streptomyces sp. A12 were challenged to an adaptation schedule of successive cultures on a fresh solid medium supplemented with PAHs, decreasing concentration of glucose in each step. As a result, an enhanced degradation of PAHs by adapted strains was observed in the presence of glucose as co-substrate, without degradation of phenanthrene and pyrene in MM while an increase to up to 50% of degradation was seen with these strains in glucose amended media. An internal fragment of the catA gene, which codes for catechol 1,2-dioxygenase, was amplified from both Rhodococcus strains, showing the potential for degradation of aromatic compounds via salycilate. These results allow us to propose the usefulness of these actinomycete strains for PAH bioremediation in the environment.